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Non-Canonical Photodynamics of the Orange/Green Cyanobacteriochrome Power Sensor NpF2164g7 from the PtxD Phototaxis Regulator of Nostoc punctiforme Julia Kirpich, Che-Wei Chang, Dorte Madsen, Sean Marc Gottlieb, Shelley S. Martin, Nathan Clarke Rockwell, J. Clark Lagarias, and Delmar S. Larsen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01252 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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Biochemistry

Non-Canonical Photodynamics of the Orange/Green Cyanobacteriochrome Power Sensor NpF2164g7 from the PtxD Phototaxis Regulator of Nostoc punctiforme

Julia S. Kirpich,a# Che-Wei Chang,a# Dorte Madsen,a Sean M. Gottlieb,a Shelley S. Martin,b Nathan C. Rockwell,b J. Clark Lagarias,b and Delmar S. Larsena*

a

Department of Chemistry University of California, Davis One Shields Ave, Davis, 95616 b

Department of Molecular and Cell Biology University of California, Davis One Shields Ave, Davis, CA, 95616

* Corresponding Author: [email protected] #

These authors contributed equally to this manuscript. 1 ACS Paragon Plus Environment

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Abbreviations used in this paper: CBCR, cyanobacteriochrome; EADS, evolution-associated

difference spectrum; SADS, species-associated difference spectra; LADS, lifetime-associated difference spectra; DADS, decay-associated difference spectra; ESI, excited-state intermediate; ESA, excited-state absorption; SE, stimulated emission; GSB, ground-state bleach; GAF, domain name derived from cGMP phosphodiesterase/adenylyl cyclase/FhlA; GSA, ground-state absorbance;

PAS,

(Per-ARNT-Sim);

PHY,

Phytochrome

phycocyanobilin; NOPA, non-collinear optical parametric amplifier; state of red/green CBCRs;

15E

specific

domains;

PCB,

15Z

Pr, red-absorbing ground

Pg, green-absorbing photoproduct state of red/green CBCRs; Pg*,

excited-state population(s) derived from photoexcitation of Pg or quantum yield; ΦL, quantum yield of generating Lumi photoproduct.

2 ACS Paragon Plus Environment

15E

Pg; Φ, total photocycle

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Biochemistry

ABSTRACT Forward and reverse primary (10 ns) photodynamics of cyanobacteriochrome (CBCR) NpF2164g7 were characterized by global analysis of ultrafast broadband transient absorption measurements. NpF2164g7 is the most C-terminal bilin-binding GAF domain in the Nostoc punctiforme phototaxis sensor PtxD (locus Npun_F2164). Although a member of the canonical red/green CBCR subfamily phylogenetically, NpF2164g7 exhibits an orange-absorbing 15ZPo dark state instead of the typical red-absorbing 15ZPr dark-adapted state of this subfamily. The green-absorbing 15EPg photoproduct of NpF2164g7 is unstable, allowing this CBCR domain to function as a power sensor. Photoexcitation of the

15Z

Po state triggers

inhomogeneous excited-state dynamics, with three spectrally and temporally distinguishable pathways, to generate the light-adapted 15EPg state in high yield (estimated at 25-30%). Although observed in other CBCR domains, the inhomogeneity in NpF2164g7 extends far into secondary relaxation dynamics (10 ns -1 ms) through to formation of primary dynamics after photoexcitation of

15E

Pg. In the reverse direction, the

15E

Pg are qualitatively similar to those of other

red/green CBCRs, but secondary dynamics involve a "pre-equilibrium" step before regenerating 15Z

Po. The anomalous photodynamics of NpF2164g7 may reflect an evolutionary adaptation of

CBCR sensors that function as broadband light intensity sensors.


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INTRODUCTION The development of genetically encoded biological materials to allow externally controlled light-initiated activities holds great potential to radically change the study of nature at the cellular level.1-6 Such optogenetic tools are typically designed using either "bottom-up" rational design approaches (starting with non-photoactivated components) or "top-down" approaches (starting with systems optimized for light activation).7-9 A key aspect of the latter approach is the use of existing photoreceptor proteins that have already evolved to initiate light signaling pathways. Effective engineering of photoreceptors as optogenetic reagents requires a detailed molecular understanding of the mechanisms that couple their light activation to biological function, including characterization of transient light-induced dynamics. Photoreceptors such as phytochromes have attracted interest for such bioengineering efforts. Found in plants, fungi, algae, and bacteria, phytochromes use covalently attached linear tetrapyrrole (bilin) chromophores (Figure 1A) to switch between dark-adapted red-absorbing and far-red-absorbing photoproduct states.10-13 Light excitation triggers photoisomerization of the 15,16-double bond of the bilin to yield a primary 'Lumi' photoproduct, typically on a picosecond or sub-picosecond timescale. This primary photoproduct then evolves via a series of transient intermediates over a nanosecond to millisecond timescale to generate a stable photoproduct that usually mediates the biological response.10 Red/far-red phytochromes11,

13

are multi-domain

proteins with a conserved photosensory core module typically including PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA), and PHY (Phytochrome specific) domains. Most red/far-red phytochromes require the presence of all three domains to exhibit reversible photodynamics comparable to the full length protein.14


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Biochemistry

Figure 1: (A) The molecular structure of the phycocyanobilin (PCB) chromophore in the Z (left) and proposed 'twisted' E (right) conformations. (B) Static absorption spectra of the NpF2164g7 power sensor in the dark-adapted terminal light-adapted

15E

15Z

Po state (orange curve) and the

Pg photoproduct (green curve; estimated by subtracting the

dark-adapted spectrum from the light-adapted spectrum under constant illumination). Cyanobacteriochromes (CBCRs) are distantly related cyanobacterial photoreceptors requiring only the bilin-binding GAF domain and exhibiting spectral sensitivity spanning the near ultraviolet to the near infrared using similar covalently attached bilin chromophores.15-22 Unlike most phytochromes, isolated CBCR GAF domains exhibit reversible photoswitching activities similar to those observed in the context of the full-length protein.23-25 CBCR domains are found in diverse structural contexts and often occur in tandem arrays within a single protein.26 The Nostoc punctiforme PtxD locus, Npun_F2164, abbreviated here as NpF2164,18,27,28


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is an extreme example of such an array, having a diverged N-terminal GAF domain (NpF2164g1) that does not bind chromophore followed by six CBCR domains (NpF2164g2-7) in tandem and a C-terminal methyl-accepting chemotaxis (MCP) output regulatory domain (Scheme 1). Consistent with regulation of its C-terminal MCP domain by light, PtxD has been shown to be essential for phototaxis of N. punctiforme hormogonia.28

Scheme 1: Domain structure of the full-length PtxD protein including the methylaccepting chemotaxis (MCP) output regulatory domain. Each GAF domain is color coded to the peak absorption spectra of their dark-adapted (left) and light-adapted (right) states. Each individual GAF domain is numbered to indicate its location in PtxD. NpF2164g7 is the 7th domain in the PtxD domain structure, photoswitching between orange- and green absorbing states. Individual CBCR GAF domains of PtxD exhibit a wide range of spectrally and kinetically distinct photoswitching activities that reflect differences in amino acid residues responsible for tuning the embedded phycocyanobilin (PCB) chromophore absorption spectrum. For example, NpF2164g2 and NpF2164g3 belong to the insert-Cys CBCR subfamily and exhibit ultraviolet/blue (UV/blue) and violet/orange photocycles, respectively.16 The other four domains belong to the canonical red/green CBCR subfamily.26 Such CBCRs typically switch between red-absorbing (15ZPr) dark states and green-absorbing (15EPg) light states.18 NpF2164g4 and NpF2164g6 both exhibit red/green photocycles. However, the green-absorbing

15E

Pg

photoproduct of NpF2164g4 is considerably more stable than that of NpF2164g6, with the 6 ACS Paragon Plus Environment

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Biochemistry

NpF2164g6 photoproduct rapidly reverting to the red-absorbing

15Z

Pr dark state in darkness.18

The CBCR domain immediately adjacent to the “output” MCP domain, NpF2164g7, is thought to be the most important for modulating signaling activity by analogy to other photoreceptor systems.1 NpF2164g7 also is a member of the red/green CBCR lineage phylogenetically; however, instead of the red-absorbing dark state (λmax ~ 650 nm) found in most members of this lineage,18,

27, 29-33

NpF2164g7 exhibits an orange-absorbing

15Z

Po dark state (λmax = 608 nm)

(Figure 1B; orange curve). The light-adapted 15EPg state of NpF2164g7 (green curve) is similar to the light-adapted states observed in other red/green CBCRs characterized to date.24,

29-32, 34, 35

However, this photoproduct state reverts back to the orange-absorbing 15ZPo dark state so rapidly that green light illumination does not accelerate its regeneration. NpF2164g7 thus is able to provide a linear response to light intensity across a broad spectral range (ca. 300-620 nm) and therefore can function as a “power sensor”.18 Similarly fast dark reversion has been reported for other members of this subfamily36, 37 as well as for one member of the more recently described DXCIP subfamily.38 Characterization of NpF2164g7 could provide a paradigm for analysis of photodynamic behavior in a broad range of CBCR power sensors as they become available. The photocycles of CBCRs and phytochromes can be modeled using the sequential photodynamic pathway depicted in Scheme 2 (central 'Yin-yang' inset). For the forward reaction, photoexcitation of the dark-adapted state (15ZPo for NpF2164g7 or

15Z

Pr for other red/green

CBCRs in the family) initially generates a mixture of excited-state 15ZPo* species, one or more of which decay on an ultrafast timescale to yield a red-shifted ground-state Lumi photointermediate, i.e., Lumi-O. The Lumi intermediate(s) subsequently evolve(s) through (a) Meta intermediate(s) (e.g., Meta-O1, Meta-O2) to generate the light-adapted state (15EPg in NpF2164g7). Furthermore, the number, spectra and kinetics of the intermediates varies from


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system to system and, in some cases, these intermediates do not always evolve sequential as Scheme 2 suggests. The light-adapted state is metastable and can evolve back to the darkadapted state thermally or via light illumination, When the light-adapted state is photoexcited, the reverse photodynamics follow an anologous sequence of events to form the dark-adapted state.

Scheme 2. A simplified conceptual photocycle for CBCRs and phytochromes involves photoconversion between a dark-adapted state (15ZPo for NpF2164g7) and a photoproduct state (15EPg for NpF2164g7). Dynamics observed during the first 10 ns after photoexcitation of either state are primary dynamics involving both excited-state evolution and formation of the ground-state Lumi intermediates. Secondary dynamics then track the evolution of the Lumi through multiple Meta intermediates to generate the terminal state. The number of Meta intermediates and their kinetic and spectral properties depend on the nature of the sample.


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Biochemistry

The dynamics of the NpF2164g4, NpF2164g5, and NpF2164g6 domains from PtxD have previously been characterized on the ultrafast timescale, demonstrating that primary photoconversion pathways of red/green CBCRs are generally conserved.33 The violet/orange photocycle of NpF2164g3 also has been studied on a broader timescale from fs to ms, providing the first characterization of light-initiated dynamics in the insert-Cys subfamily.39 However, time-resolved characterization of red/green CBCRs to date has been limited to domains that have red-absorbing dark states.29, 31, 33, 40-42 Here we characterize the primary (10 ns) photodynamics for both forward (15ZPo → Pg) and reverse (15EPg →

15Z

Po) reactions of

the isolated NpF2164g7 domain using broadband transient absorption (TA) spectroscopy (Scheme 2). Our studies provide the first characterization of any CBCR-based power sensor and reveal novel aspects of NpF2164g7 photodynamics that may reflect evolutionary adaptation for function as a broadband light intensity sensor.

EXPERIMENTAL Protein Expression, Purification, and Characterization. NpF2164g7 was expressed and purified as previously described.18, 40 Static absorption spectra of 15ZPo and 15EPg were acquired at 25°C on a Cary 50 spectrophotometer modified to allow top-down illumination of the sample to switch between the two photostates.43 The primary and secondary signals were measured at least three times to ensure reproducibility and the datasets with the best signal to noise were analyzed and discussed below. The details of the apparatus used to measure the primary (100 fs to ~8 ns) dynamics of both forward and reverse reactions have been published previously (Figure S1A).44 The features relevant to the data reported here are briefly discussed below. A 800-nm Ti:Sapphire amplified laser (SpectraPhysics Spitfire Pro) pumped two separate homebuilt non-collinear optical 9 ACS Paragon Plus Environment

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parametric amplifiers to generate the excitation pulses used for initiating the forward (590 nm at 350 nJ) and reverse (515 nm at 330 nJ) photodynamics. These wavelengths were selected to be near the peaks of the static absorption spectra of

15Z

Po and

15E

Pg (Figure 1B). The instrument

response functions for both directions were estimated at ~120 fs from the signal rise times of the excited-state absorption (ESA) bands. The instrumental setup for characterizing secondary dynamics is based on a similar design to the apparatus used to characterize the primary dynamics.39 The broadband probe pulses were generated by focusing a portion (~1 µJ) of the 800-nm Ti:Sapphire amplified pulses into a slowly translating 10-mm thick CaF2 crystal to generate a continuum that spans the full visible range. After sample interrogation, these probe pulses were imaged via a spectrograph (Oriel Instruments 77480) onto a 512-pixel silicon diode array (Hamamatsu C7884-8L003). The primary difference between primary and secondary experimental setups is that excitation pulses for initiating the secondary signals originate from an independent Q-switched diode-pumped solid-state YAG laser (Pulselas-A-532-300) with 15-20 µJ per pulse at 1 kHz and pulse width of 500 ps (Figure S1B). The 532-nm excitation pulses for initiating the secondary photochemistry in both directions were generated via second harmonic generation of the YAG’s 1064-nm fundamental light with a 5-mm thick potassium dihydrogen phosphate crystal. This 532-nm pump pulse is spectrally resonant with the weakly resonant with the

15E

Pg spectrum of NpF2164g7 (Figure 1B, green curve), but is also

15Z

Po spectrum (orange curve). Consequently, the raw signals of

NpF2164g7 initiated from 532-nm excitation contain contributions from both forward (15ZPo → 15E

Pg) and reverse (15EPg →

15Z

Po) reaction signals depending on the presence of

sample. Clean forward reaction signals are extracted by subtracting the pure


15E

15E

Pg in the

Pg signals from

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Biochemistry

the measured signals after being scaled to remove signatures of the

15E

Pg bleach. The temporal

resolution of the secondary experiments was estimated at 550 ps, based off the rise time of the GSB signals. All measurements were performed at room temperature (25 °C). For measurement of primary photodynamics, the sample was flowed continuously (~20 mL/min) in a closed circuit to ensure a fresh sample for each excitation pulse. For the secondary measurements, the sample was circulated at a slower rate (~10 mL/min) to ensure that excited sample did not flow out of the excitation volume region and alter the measured signals within the 1-ms probe timescale. The sample was flowed through a 2-mm thick quartz cuvette with an optical density of 0.3-0.4 of the dark-adapted

15Z

Po state (at that pathlength). For the reverse

reaction measurements (both primary and secondary), the sample was continuously illuminated with a red CW laser diode (630 nm, 20 mW) immediately surrounding the probe region to enrich the 15EPg state sufficiently for interrogation. As an independent check on secondary photodynamic measurements, 1-ms transient spectra for both reaction directions were also measured for the primary datasets by shifting the 1kHz probe pulse train slightly before (~1 ps) the pump pulse. This results in the collection of a transient spectrum initiated by the previous pump pulse. To facilitate this, the sample flow rate was reduced to retain sample within the excitation laser volume in the flow cell. Under normal flow conditions, the “pre-time zero” (1 ms) difference spectrum was negligible. Subsequent dynamics (e.g. at 4 ms or 6 ms) do not contribute to the measured signals, since the flow rate completely refreshes the sample within the excitation volume on this timescale. Global Analysis. Broadband signals were analyzed by multi-wavelength global analysis routines that “globally” fit the data to an underlying model of interconnected and evolving populations.45, 46

The central goal of this analysis is to decompose the measured signals into an admixture of


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transient populations with time-independent spectra and time-dependent amplitudes to resolve the underlying dynamics. This is accomplished by fitting the data to a postulated system of linear first-order differential equations (Equation 1):

= + ∑

(1)

where ni is the ith microscopic population, Ai is its initial occupancy, I(t) is the pump pulse profile, and Kij are elements of the rate constant matrix that describe the exponential evolution from one population to another. Two limiting models are commonly used to interpret broadband TA data: an exclusively sequential approach or an exclusively parallel approach. Within the sequential model (A→ B→ C→ D…), the first population (A) is photo-generated by the laser pulse and evolves directly into the second population (B) that then evolves into the third population (C); the model continues until the chain is completed. This sequential evolution is typically modeled with no loss of amplitude and with successively slower timescales; the spectra extracted from this limiting model are referred to as Evolution Associated Difference Spectra (EADS). Within the parallel model, all populations are generated after photoexcitation and then independently decay (but not into new populations) with different timescales. This model is the global analysis version of fitting transient data to a “simple sum of exponentials” that is commonly used to fit transient signals. The spectra extracted from this limiting model are called Decay Associated Difference Spectra (DADS) or Lifetime Associated Difference Spectra (LADS). Both limiting models provide a good starting point for interpreting the measured TA data, but often the photodynamics of photobiological systems follow neither limiting model and require the construction of more complex, system-specific “target models” to interpret the data.33, 39-42, 47, 48

Target models are best constructed with secondary a priori knowledge or with guidance from


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Biochemistry

established photochemical principles subject to fitting the data with unstructured and minimized residuals. The resulting spectra are termed Species Associated Difference Spectra (SADS) if and only if the target model accurately represents the true photoinduced dynamics of the system. If not, the extracted spectra are mixtures of the underlying SADS and are of reduced utility in interpreting the measured data.29, 40, 41, 47, 49

RESULTS Forward Reaction Dynamics (15ZPo → after photoexcitation of

15E

Pg). Transient spectra of NpF2164g7 (200 fs - 6 ns)

15Z

Po are shown in Figure 2A. The early-time spectrum (black curve) is

ascribed to the excited-state 15ZPo* population with broad positive excited-state absorption (ESA) bands on the high (< 530 nm) and low-energy (> 640 nm) sides of the negative Po bleach. As expected, this ground-state bleach (GSB) resembles the NpF2164g7, the low-energy ESA band of

15Z

Po spectrum (unfilled circles). In

15Z

Po* completely obscures the negative stimulated-

emission (SE) band typically observed in red/green CBCRs,29, 33, 40.As

15Z

Po* decays, a positive

peak near 650 nm develops. We attribute this absorption to the red-absorbing primary photoproduct Lumi-Or. This population does not exhibit further evolution within the 7 ns time range of the primary dynamics dataset. The 15ZPo* to Lumi-Or dynamics are better resolved in the kinetic traces of Figure 2B, which reveal multi-phasic decay kinetics of

15Z

Po* extending from 1

ps to 100 ps. In other CBCRs (and phytochromes), we interpreted such multi-exponential dynamics as arising from sample inhomogeneity with multiple co-existing excited-state populations generated from multiple ground-state populations.33, 39-41, approach in the analysis and interpretation of these data (vide infra).


47, 50

We adopt a similar

Biochemistry

500

550

600

650

Probe Times (ps) 700 0

1

10

100

1000 5000 450 nm 620 nm 15 670 nm

A. 0

0 4

-15

200 fs 1 ps 10 ps 100 ps -30 6 ns

C. 4 2

B.

1000

5000

-15

2 0

-30 550 nm 620 nm 650 nm

1 ns 100 ns 1 µs 300 µs 1 ms

D. 4 2

0

0

-2

∆ A (mOD)

∆ A (mOD)

15

∆ A (mOD)

Wavelength (nm) 450

∆ A (mOD)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 40

-2

450

500

550

600

650

700 0.00

0.01

Wavelength (nm)

0.1

1

10

100

1000

Probe Times (µs)

Figure 2: Primary (< 10 ns) and secondary (> 10 ns) forward (15ZPo →

15E

Pg) reaction

photodynamics of NpF2164g7. (A) Primary and (C) secondary spectra at select probe times with overlapping inverted 15ZPo spectrum and

15E

Pg-15ZPo spectrum (unfilled circles

in A and C, respectively); (B) primary and (D) secondary kinetics at select probe wavelengths. The kinetics were fit (solid lines in B and D) with the respective target models shown in Figures 3 and 4. Primary dynamics between 50 ps to 7 ns are shown with a linear scale in the inset of panel B.

Transient spectra for the forward secondary photodynamics (1 ns - 1 ms) are presented in Figure 2C. The Lumi-Or photoproduct observed in the primary signals (Figure 2C; black curve) decays on a ~200 ns timescale to form a new green-absorbing species absorbing at 540 nm (green and magenta curves) although a sub-population of Lumi-Or persists beyond 1 ms. The terminal 1-ms spectrum (magenta curve) matches the

15E

Pg -

15Z

Po difference spectrum (unfilled

circles) at higher energies (< 610 nm), deviating slightly at lower energies due to a residual long14 ACS Paragon Plus Environment

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Biochemistry

living Lumi-Or population. These data indicate that the light-adapted

15E

Pg state is formed on

multiple timescales via multiple Lumi-Or populations (discussed below). This multiphasic evolution can be observed in the 650-nm kinetics trace in Figure 2D (dark red curve), which directly tracks the Lumi-Or population decay kinetics over multiple timescales, and can also be seen in the GSB kinetics at 620 nm (red curve) and in the growth of the

15E

Pg population at 550

nm (green curve). For initial analysis of the primary data, a sequential EADS analysis with five populations was necessary to fully described the primary forward reaction data with 68 fs, 540 fs, 7.5 ps, 120 ps, and ∞ timescales (Figure S2 and S3). The initial 68-fs phase comprises a mixture of crossphase modulation,51, 52 stimulated Raman scattering,53 and multi-photon absorption signals,7, 54 which are collectively ascribed to a general “coherent artifact”.55-57 The other four phases represent the dynamics of true microscopic populations that correspond to three decaying

15Z

Po*

populations (characterized by the high energy ESA bands) and a growing Lumi-Or population. To more explicitly describe the independent evolution of these four populations, an inhomogeneous target model was constructed with guidance from the EADS analysis (Figure 3A) that was consistent with the sequential EADS model as confirmed by similar residuals (Figure S3). This model postulates that three co-existing ground-state photoexcited by the pump pulse to generate three excited-state independently into Lumi-Or or ground state. Each

15Z

Po populations are

15Z

Po* populations that decay

15Z

Po* sub-population is assigned a unique

photoproduct quantum yield (ΦL) and apparent decay timescale for generating Lumi-Or or ground state (Table 1). The EADS derived from the sequential analysis (Figure S2) suggests that the fastest decaying 15ZPo* population (~1.5 ps) does not generate significant Lumi-Or (based on the similarity of EADS2 and EADS3 (Figure S2C)). Similar to the forward reaction dynamics of


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all measured red/green CBCRs to date,29, 33, 40, 41 the slower 15ZPo* populations appear responsible for generating Lumi-Or and propagating the photocycle (Table 1). Although the secondary photodynamic data reveals three distinct photoproduct populations (Lumi-Or I, Lumi-Or II, and Lumi-Or III: see below), they are modeled as a single Lumi-Or population in the analysis of the primary data because they exhibit near-identical spectra.

Figure 3: Target-based global analysis for the primary (< 10 ns) forward reaction (15ZPo →

15E

Pg) dynamics of NpF2164g7: (A) target model; (B) SADS; (C) concentration

profiles. Unfilled circles in panel B represent the inverted

15Z

Po ground-state absorption

spectrum. Residuals of the fitted model are shown in Figure S3 and Table S1.


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Biochemistry

Table 1: Target analysis parameters for the primary forward reaction (15ZPo → 15EPg) Primary photodynamics (< 10 ns) ESI 1 (35 %)

ESI 2 (55 %)

ESI 3 (10 %)

1.5 ps

12 ps

220 ps

τapparent τbranched Φbranching ΦL

→ GS 1.5 ps 100 %

→ Lumi-Or -

→ GS → Lumi-Or 18 ps 35 ps 66 % 34 % 25-30 %

→ GS 1 ns 22 %

→ Lumi-Or 280 ps 78 %

Secondary photodynamics (> 10 ns) Lumi-Or I (34 %) → Meta-Og I τ

τ

120 ns Meta-Og I → Pg 1.4 μs

Lumi-Or II (33 %)

Lumi-Or III (33 %)

→ Meta-Og II

→ Meta-Og III

1.5 μs Meta-Og II → Pg 550 μs

1.4 ms Meta-Og III → Pg > 1 ms

An important, but often difficult, feature of the target analysis is the estimation of the absolute quantum yield (ΦL) for generating Lumi-Or. Previously, ΦL values were estimated by comparison to reference samples with established ΦL values29,

33

or by the use of three-pulse

pump-dump-probe techniques.42 However, NpF2164g7 is the first orange/green CBCR domain to be studied with transient techniques, so a reference sample with similar spectral properties and a known ΦL is lacking.33, 39, 50, 58, 59 For modeling NpF2164g7 dynamics, the total Lumi-Or yield was estimated at 25-30% via three complementary, albeit indirect, approaches: (A) Our previous study of red/green CBCR domains demonstrated a rough correlation between the slowest excited-state decay constant and ΦL.33 Applying this relationship to NpF2164g7 predicts a ΦL between 20% and 40% based on the slowest excited-state decay constant (220 ps).


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(B) The dark-adapted adapted

15E

15Z

Po absorption spectrum of NpF2164g7 strongly resembles the light-

Po state of the violet/orange CBCR NpF2164g3 (Figure S4A, NpF2164g7:

608 nm; NpF2164g3: 588 nm).16,

39, 60

Despite the different geometries of the PCB

chromophore (15E vs. 15Z configurations), the measured TA data also exhibit similar spectral features in these two samples (Figure S5). The initial spectra of both CBCRs immediately after excitation exhibit strong ESA bands from 450-525 nm and from 650710 nm, respectively and both exhibit GSB at ~575-640 nm (Figure S4C, solid lines: 150-fs TA spectra). Both spectra also have similar amplitude for spectra and amplitude (with respect to the initial excited-state spectrum) of Lumi-Or species. Since ΦL in NpF2164g3 was estimated at ~26%, the Lumi-Or yield of NpF2164g7 is also estimated at ~ 26%. However, it should be noted that error of estimating of ΦL in NpF2164g3 was not insignificant, since no suitable reference CBCR or phytochrome with a known ΦL exists for comparison. (C) For several CBCRs with well-resolved broadband TA signals, we find that the SADS amplitude of the primary photoproduct is comparable to the amplitude of the red-most ESA band of the excited-state populations.33, 39-41 While indirect and empirical, this is a consistent feature of primary dynamics in red/green CBCRs. For NpF2164g7, this correlation of spectral signatures corresponds to ΦL between 25-30% (Figure 3B). These three approaches are admittedly indirect assessments of the forward ΦL, but they are selfconsistent in their estimation of a ΦL that varies between 25% and 30%. The fastest decaying excited-state

15Z

Po* population in the proposed target model (ESI1:

35% of the total) decays with an apparent (i.e., observed) 1.5-ps lifetime and does not generate a measurable Lumi-Or population. This assessment is based on the difference of the scaled EADS2


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Biochemistry

and EADS3 spectra, which shows only loss of excited-state population and no apparent Lumi-Or features (Figure S2C). The other two 15ZPo* populations (ESI2: 55%; ESI3: 10%) generate LumiOr and decay with 12 ps and 220 ps lifetimes, respectively. The SADS for these three

15Z

Po*

populations exhibit similar qualitative features including ESA and GSB bands (Figure 3). However, these three SADS are sufficiently different in the long wavelength region that assigning them to a single species in the analysis significantly reduces the quality of the fit (not shown). Hence, the inhomogeneity in the three 15ZPo* populations is both spectral and temporal, which is atypical for CBCR and phytochrome systems.33, 40, 49 To commence analysis of secondary dynamics, we compared datasets from primary and secondary timescales. We noted that the 1-ms spectrum of the forward reaction extracted from the primary measurements (Figure S6A) exhibits two positive bands at ~640 nm (from Lumi-Or) and ~530 nm (from the subsequent Meta-Og intermediate), in agreement with the 1-ms spectrum extracted from the secondary measurements (Figure 2C). These 1-ms spectra match neither the spectrum for Lumi-Or formation nor that for full photoconversion (Fig. S5A). Therefore, the Lumi-Or to Meta-Og evolution is incomplete within 1 ms, in agreement with the ongoing evolution observed at late times in the secondary data (Figure 2D). Secondary data therefore could be fitted by a sequential model having 4 compartments connected by 3 timescales (Figure S7). Within this analysis, EADS show clear persistent and multiphasic relaxation kinetics of the Lumi-Or mixture (Figure S7B). The coexistence of both Meta-Og and Lumi-Or and the multiphasic evolution observed in Figure 2D necessitate adoption of a more complex target model with multiple co-existing Lumi-Or sub-populations. We designate Lumi-Or I, Lumi-Or II, and Lumi-Or III sub-populations within our target model, with corresponding lifetimes of 120 ns, 1.5 µs, and 1.4 ms, respectively (Table 1). At later times, we observed similar kinetic


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inhomogeneity causing us to designate three spectrally identical Meta-Og sub-populations (MetaOg I, Meta-Og II, and Meta-Og III) that generate

15E

Pg with lifetimes of 1.4 µs, 550 µs, and >1

ms, respectively (Table 1). An alternative homogeneous pre-equilibrium model can be postulated with the Lumi-Or and Meta-Og populations “pre-equilibrating” before forming the terminal

15E

Pg

state (Figure S8A). While admittedly simpler than the inhomogeneous model in Figure 4, this model predicts a terminal

15E

Pg -

15Z

Po difference spectrum (Figure S8B; blue curve) that

overestimates the relative amplitude of

15Z

Po. Since this aspect is better described by the

proposed inhomogeneous model (Figure 4B & S8E), we argue that the inhomogeneous model is a more accurate description of the underlying forward photodynamics.


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Biochemistry

Figure 4: Target-based global analysis for the secondary (> 10 ns) forward (15ZPo → 15E

Pg) dynamics: (A) target model; (B) SADS; (C) concentration profiles. The fit of this

model to the data can be evaluated in Figure 2D. Unfilled circles in panel B, static 15EPg 15Z

Po spectrum. Dashed and dotted lines in Figure 4C represent concentrations of reactive

intermediates of II and III populations, correspondingly. Residuals of the model fit are contrasted in Figure S9 and Table S1.

Reverse Reaction Dynamics (15EPg → exciting

15E

15Z

Po). The reverse reaction dynamics initiated by

Pg exhibit similarities to the forward reaction dynamics, but with several key

differences. The initial excited-state

15E

Pg* spectrum (Figure 5A; black spectrum) exhibits two

ESA bands near 450 nm (weak) and 680 nm (strong). In contrast to the 21 ACS Paragon Plus Environment

15Z

Po* spectrum (Figure

Biochemistry

2A; black spectrum), the

15E

Pg* spectrum exhibits a resolvable SE band at 600 nm. The GSB

matches the ground-state 15EPg spectrum (unfilled circles). As 15EPg* decays, the primary orangeabsorbing Lumi-Go photoproduct appears, peaking near 610 nm (green curve). No subsequent evolution is observed within the 7-ns temporal limit of the ultrafast experiment. As with the forward reaction dynamics (Figure 2B), the excited-state decay kinetics of the reverse dynamics are also multiphasic, albeit with slower timescales for the productive, long-lived populations (Figure 5B and see below).

∆ A (mOD)

A.

450 5

500

550

600

650

Probe Times (ps) 700

0

1

10

100

1000 5000 450 nm 10 550 nm 620 nm

0

5 0

-5

2000 4000 6000

200 fs 1 ps 100 ps 1 ns 6 ns

-10

2 0 -2

C.

550 nm

620 nm(x1.5)

B.

-5 -10

∆ A (mOD)

Wavelength (nm)

-15

D.

650 nm

2

2 500

600

700

0

0 -2

1 ns 100 ns 3 µs 20 µs 1 ms

-2 450

500

550

600

650

∆ A (mOD)

4

∆ A (mOD)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 40

-4

700 0.00

0.01

0.1

Wavelength (nm)

1

10

100

1000

Probe Times (µs)

Figure 5: Primary (< 10 ns) and secondary (> 10 ns) reverse (15EPg →

15Z

Po) reaction

photodynamics of NpF2164g7. (A) Primary and (C) secondary spectra at selected probe times with overlapping inverted 15EPg spectrum and

15Z

Po-15EPg spectrum (unfilled circles

in A and C (inset), respectively); (B) primary and (D) secondary kinetics at select probe wavelengths. The data were fit (solid lines in B and D) with the target models in Figures 6 and 7. The kinetics of the primary dynamics between 1 to 7 ns are presented in detail in the inset of panel B. The 1 ms spectrum is compared to the static difference spectrum in the inset of panel C. 22 ACS Paragon Plus Environment

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Biochemistry

The secondary reverse reaction signals (Figure 5C and D) exhibit more complex behavior than the secondary forward dynamics. As expected, the Lumi-Go spectrum observed in the primary dynamics is well characterized at 1 ns with a peak at ~600 nm (Figure 5C; green curve). Subsequent evolution involves a small amplitude blue shift to 590 nm (Figure 5C; red curve), which we ascribe to the Meta-Go state. By 20 µs, a far-red-absorbing Meta-Gf state with a peak at ~675 nm (Figure 5C; black curve) is apparent, which ultimately converts to 15ZPo (Figure 5C; inset). These spectral dynamics occur non-monotonically, with Lumi-Go to Meta-Go evolution occurring on a ~200 ns timescale (Figure 5D; red curve) and with growth and decay of Meta-Gf on 50 µs and 100 µs timescales, respectively (Figure 5D; dark red and green curves). The growth of 15ZPo is observed on a ~500 µs timescale. A five-population sequential model (Figure S10) captures the full dynamics of the primary reverse dynamics, with time constants of 80 fs, 2.2 ps, 73 ps, 530 ps, and ∞ (stable terminal state on this timescale). The first phase corresponds to the same coherent artifact observed in the forward reaction and the other four states are ascribed to three

15E

Pg* sub-

populations that decay to the primary Lumi-Go photoproduct (Figure 6A). In contrast to the forward reaction dynamics, in which each excited-state population exhibiting slightly different 15Z

Po* spectra (Figure 3B), the three 15EPg* populations share the same spectrum (Figure 6B, black

curve). These spectra were thus locked together for convenience in fitting. Unlocking the SADS did not appreciably alter the fit or ESI spectra (data not shown).


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6: Target-based global analysis for the primary (< 10 ns) reverse (15EPg →

Page 24 of 40

15Z

Po)

reaction dynamics: (A) target model; (B) SADS; (C) concentration profile. Unfilled circles in panel B represent the inverted

15E

Pg ground-state absorption spectrum (Figure

1B). Dashed and dotted lines in Figure 6C represent concentrations of reactive intermediates of II and III populations, correspondingly.

The absolute Lumi-Go ΦL used in the target model (Figure 6) was estimated at ~43% via comparison to the primary reverse photodynamics of the red/green CBCR NpF2164g6 (Figure S11), which has an established ΦL of 40% using the pump-dump-probe technique.29 The initial NpF2164g7 spectrum (quantifying the Pg* population amplitude) was scaled to match the ESA band of NpF2164g6 at ~450 nm, as that region represents relatively clean excited-state signals in the transient absorption spectrum (Figure S4D).29 The same scaling factor was then applied to 24 ACS Paragon Plus Environment

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Biochemistry

the terminal Lumi-Go photoproduct spectrum at later times. The amplitude of the Lumi-Go spectra of the two samples exhibited comparable amplitudes (when scaled to the same Pg* population), so they have comparable ΦL values. This value was optimized to ~43 ± 5% in the global analysis (Table 2).

Table 2: Target analysis parameters for the reverse reaction (15EPg → 15ZPo) Primary photodynamics (< 10 ns) ESI 1 (5 %)

ESI 2 (36 %)

ESI 3 (59 %)

0.6 ps

15 ps

280 ps

τapparent τbranched Φbranching ΦL

→ GS 0.6 ps 100 %

→ Lumi-Go -

→ GS 650 ps 2%

→ Lumi-Go 15 ps 98 % ~43 %

→ GS 320 ps 88 %

→ Lumi-Go 2.3 ns 12 %

Secondary photodynamics (> 10 ns) 46 µs

Lumi-Go → Meta-Go τ τ

Meta-Gf

Meta-Go 55 µs

180 ns Meta-Go → Po 420 μs

A sequential EADS analysis of the secondary data was used to characterize the relaxation dynamics occurring, identifying four timescales with three time constants (Figure 7A). The EADS extracted from this analysis (Figure 7B) predicts a terminal spectrum (EADS4; blue curve) with two positive bands at 575-710 nm. This “double bump” feature is likely to be the result of mixing two (or more) SADS. Hence, the underlying dynamics are probably more complicated than the sequential, irreversible dynamics of the EADS model. We therefore evaluated a “pre-equilibrium” equilibrium target model similar to that rejected for the secondary 25 ACS Paragon Plus Environment

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Page 26 of 40

forward dynamics (Figure S8). The pre-equilibrium model for the reverse reaction (Figure 7D) postulates that Lumi-Go evolves directly into an orange-absorbing Meta-Go state peaking at 595 nm on a 180-ns timescale. Meta-Go is then envisaged to establish an equilibrium on a 46 µs timescale with a red-shifted Meta-Gf population (blue curve) peaking at 675 nm. Subsequent evolution of this equilibrated mixture is tracked in the far-red kinetics (Figure 5D; dark red curve) and matches formation of the 15ZPo state (Figure 5D; green curve). This occurs on a 420µs timescale and affords a difference spectrum identical to

15Z

Po –

15E

Pg (Figure 7E; unfilled

circles).

Figure 7: Target models for the secondary (> 10 ns) reverse (15EPg →

15Z

Po) reaction

dynamics for the sequential model (A, B, C) and the equilibrium model (D, E, F) including target models (A, D) SADS (B, E); and concentration profiles (C, F). Unfilled circles in panel B and E represent the 15ZPo - 15EPg spectrum. 26 ACS Paragon Plus Environment

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Biochemistry

The inclusion of the Meta Go - Meta-Gf equilibrium removes the dubious double-bump feature in the EADS3 spectrum (Figure 7B; blue curve), which argues that the pre-equilibrium model is a more accurate description of the underlying photodynamics. Unfortunately, the rapid equilibration relative to

15Z

Po generation means that we cannot identify whether Meta-Go or

Meta-Gf is the precursor to 15ZPo; hence this is purposely left undefined in the model (Figure 7D). An alternative “detour” model exhibits similar contamination of the terminal 15ZPo spectrum with Meta-Gf features (Figure S12) and was discounted as a viable alternative for this reason.

DISCUSSION The primary and secondary transient dynamics of NpF2164g7 collected in both forward and reverse directions are summarized in Figure 8. Temporal heterogeneity is observed in the primary dynamics of both forward and reverse light-induced reactions. For the forward dynamics, this inhomogeneity is manifested by three spectrally distinct excited-state

15Z

Po

populations, the two longest living of which evolve into the primary Lumi-Og photoproduct. Lumi-Or behaves as a mixture of three spectrally indistinguishable populations that evolve to Meta-Og with 120 ns, 1.5 µs and 1.4 ms time constants. Meta-Og similarly evolves as three apparent populations with 1.4 µs, 550 µs, and >1 ms time constants to yield the terminal

15E

Pg

photoproduct state. The reverse reaction proceeds via three spectrally indistinguishable excitedstate 15EPg populations, two of which evolve into the primary Lumi-Go photoproduct. Subsequent evolution of Lumi-Go occurs on a 180 ns timescale to yield a blue-shifted Meta-Go species that equilibrates with a FR-absorbing Meta-Gf species prior to generation of the final 15ZPo dark state on a ~420 µs timescale.


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Figure 8: The complete photocycle of the NpF2164g7. The wavelengths noted are the peak positions for the estimated SADS. The half-time of dark reversion was measured at 4.1 s previously.18

NpF2164g7 belongs to the canonical red/green subfamily of CBCRs, but its spectral properties distinguish it from most CBCRs in the red/green subfamily. The

15Z

Po dark state

(peaking at 608 nm) is distinctly blue-shifted compared to other CBCRs in this subfamily, which characteristically exhibit red-absorbing

15Z

Pr dark-adapted states peaking at 650 ± 20 nm.17-19, 33

This blue shift implies that the 15ZPo chromophore may be more deformed compared with that of other red/green CBCRs, although other spectral tuning mechanisms such as deprotonation61 or hydration24 cannot be excluded a priori. This raised the question of whether the

15Z

Po state in

NpF2164g7 is twisted in such a way to be predisposed for faster and therefore more efficient excited-state isomerization than 15ZPr states of other red/green CBCRs. Our studies show that this is not the case (see below). The 15EPg photoproduct of NpF2164g7 reverts to 15ZPo at a rate of 0.2 s-1, significantly faster than typical red/green CBCRs.18 However, other members of this subfamily have been reported to exhibit comparably fast dark reversion, including AnPixJg4 and


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Biochemistry

the CBCR domain of All2699.36,37 Both proteins exhibit red-absorbing dark states, indicating that 15E

Pg state instability is not linked to a blue-shifted

15Z

Po dark state. Dark reversion of

NpF2164g7 is much faster than that of NpR6012g4, which has a stable photoproduct that lasts for days, and that of NpF2164g6, which has a less stable photoproduct lasting for minutes.18 Both of these proteins have been characterized on the ultrafast timescale,29,

41

so similar

characterization of NpF2164g7 allows us to ask whether less stable photoproducts exhibit novel photodynamics in this family of CBCRs.

Forward Dynamics (15ZPo →

15E

Pg): Gottlieb et al. identified two trends from a comparative

study of the primary forward dynamics of nine red/green CBCRs:33 (1) the more blue-shifted the ground-state absorption spectrum of a CBCR, the slower its excited-state quenching dynamics; and, (2) the more blue-shifted the ground-state absorption of a CBCR, the smaller its quantum yield in generating the primary Lumi-Rf intermediate (ΦL). Given the unique spectral and thermal recovery properties of NpF2164g7, it is not surprising that this domain does not follow either trend. NpF2164g7 exhibits an average (weighted) quenching timescale of 29 ps (Table 1), which is comparable to the fastest red/green CBCRs (observed range, 32 ps – 1.6 ns).33 Hence, while NpF2164g7 exhibits the most blue-shifted absorption spectrum of the red/green subfamily, it exhibits the fastest average excited-state quenching timescale of this subfamily. Similarly, ΦL in NpF2164g7 is estimated at 25-30% and falls at the “higher end” of the observed range for characterized red/green CBCRs (0 to 40%).29, 42 However, even this quantum yield is well below 50%, so it does not seem that the chromophore in NpF2164g7 is unusually well positioned for photoisomerization relative to other red/green CBCRs. Clearly, the factors that are responsible


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for the empirical correlation between spectral tuning and excited-state yields in red/green CBCRs are not the dominant mechanism(s) determining these processes in NpF2164g7. Forward primary and secondary photodynamics were modeled within inhomogeneous target models involving multiple co-existing sub-populations evolving simultaneously, each with different intrinsic dynamics (Figures 3A and 4A). Similar inhomogeneous models have been proposed for interpreting the primary and secondary dynamics of other CBCR domains and phytochrome proteins.29, 40, 41, 47, 49 However, the forward dynamics of NpF2164g7 are distinct from other red/green CBCRs, because the excited-state

15Z

Po* dynamics exhibit both spectral

and temporal inhomogeneity (Figure 3): each of the three excited-state 15ZPo* populations exhibit unique spectral and kinetic properties. Interestingly, the trends identified by Gottlieb et al.33 for different red/green CBCRs also extend to the observed 15ZPo* populations in NpF2164g7 (Figure 3B), with the population exhibiting the most blue-shifted bleach (blue curve) exhibiting the longer lifetime and the lower ΦL of the two active populations (Figure 3A and Table 1). A similar phenomenon was observed in the forward dynamics of the Cph1 bacterial phytochrome from Synechocystis sp.,59 which has been attributed to changes in surrounding hydrogen-bonding network rather than to structural inhomogeneity of the PCB chromophore.62 While inhomogeneity is a ubiquitous observation in the primary dynamics of CBCRs characterized to date, and indeed in the behavior of other biliproteins,63 it is rarely observed in the evolution of subsequent intermediates. That is, multiple excited-state populations typically relax into a single primary photoproduct that then behaves with apparently homogeneous, firstorder secondary photodynamics. The exception reported to date is the green/red CBCR RcaE, in which the forward (Z → E) reaction exhibits three primary photoproducts, but only one propagates to generate the terminal light-activated state.64, 65 In NpF2164g7, the inhomogeneity


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Biochemistry

observed in the excited-state dynamics uniquely extends into the resolved secondary timescales. The result of this extended heterogeneity is that three Lumi-Rf sub-populations evolve into three Meta-Og sub-populations on timescales spanning four decades in time (Figure 4A & S13). Owing to the identical spectra of the Meta-Og sub-populations, it is not possible to assign each Lumi-Rf sub-population to a corresponding Meta-Og sub-population from our analysis. The persistence of apparent parallel pathways for forward photoconversion of NpF2164g7 cannot currently be explained. Most residues predicted to be close to the photoactive D-ring of the bilin chromophore are also conserved in the red/green CBCRs AnPixJg2 and NpR6012g4, and NpR5113g2 (Table 3). The exceptions are a Gly residue in place of the conserved Ala622 on the first β strand, a Val in place of the conserved “lid” Trp655,23 a Tyr in place of the variable His659, and a Trp in place of the conserved “helix” Phe695.23 The effects of the Gly residue on spectral tuning of red/green CBCRs are unknown, but introduction of Trp for the helix Phe in NpR6012g4 did not ablate formation of

15Z

Pr.23 Similarly, Tyr is present as an

equivalent to His659 in the red/green CBCR NpR5113g2,18 and this protein exhibits a normal red/green photocycle and follows the same trends in primary photodynamics as other red/green CBCRs.18, 33 NpR5113g2 also lacks the lid Trp, but substitution of Val for Trp655 in NpR6012g4 does result in formation of a minority orange-absorbing sub-population in the 15Z state. Taken together, it is not yet clear what protein-chromophore interactions cause the unusual spectral tuning and photodynamics of NpF2164g7.


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Table 3: Comparison of amino acid residues in proximity of D-ring in chromophore pocket of NpR6012g4 and aligned NpF2164g7.18 NpR6012g4 β1: Ala622 β1: Tyr624 β2: Phe634 “lid”: Trp655 Asp-motif: His659 helix: Phe695 β5: Tyr700 β6: Tyr718

AnPixJg2 Ala Tyr Phe Trp His Phe Tyr Tyr

NpR5113g2 Ala Phe Phe Ile Tyr Phe Phe Tyr

NpF2164g7 Gly Tyr Phe Val Tyr Trp Tyr Tyr

Reverse Dynamics (15EPg→15ZPo): Despite the thermal instability of the NpF2164g7 photoproduct, the primary reverse photodynamics of NpF2164g7 are similar with those of other red/green CBCRs such as NpF2164g6 (Figures S4B & S11).29 The excited-state 15EPg spectra of both proteins exhibit clear ESA bands on both sides of the GSB and the Lumi-Go primary photoproduct peaks near 610 nm. This strong similarity suggests comparable bilin geometry and protein-bilin interactions in the two proteins. Temporally, NpF2164g7 exhibits a weighted excited-state decay constant of 170 ps (Table 1), which is slower than the timescales for E → Z isomerization in the red/green CBCRs NpR6012g4 and NpF2164g6 (from 2 ps to 37 ps).29 The quantum yield for generating the primary Lumi-Go intermediate in NpF2164g7 was estimated at ~43% (Table 2), which is very similar to the known ΦL values of NpR6012g4 and NpF2164g6 (40 9-48%).29 The observation of an equilibrium step in secondary dynamics of the reverse reaction of NpF2164g7 (Figures 7 & S14) is uncommon, but it is not inconsistent with the principle of microscopic reversibility. This principle underlies the formulation of a kinetic model for decomposing dynamics into multiple elementary processes66 and has been explicitly integrated into modeling photoactive yellow protein67,

68

and rhodopsin.45 However, microscopic


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Biochemistry

reversibility is often ignored in modeling dynamics, resulting in the reporting of effective time constants rather than “true” forward barrier crossing time constants. For the reverse photodynamics of NpF2164g7, the reversibility between separate Meta-Go and Meta-Gf intermediates is easily observed because of the strong spectral differences between these two intermediates (590 nm and 675 nm, respectively) shown in Figure 7E. Characterized red/green CBCRs typically exhibit monotonic secondary reverse dynamics, with evolution of Meta-Go (peaking around 590 nm) into

15Z

Pr (peaking around 650 nm).30, 69

This often occurs after an initial rapid Lumi-Go → Meta-Go evolution (e.g., 220 ps in NpR6012g4, with a slight blue shift).41 In NpF2164g7, non-monotonic spectral shifts may occur, because Meta-Go (peaking around 590 nm) can either evolve into 15ZPo (peaking around 608 nm) or red shift to form the far-red absorbing Meta-Gf intermediate (675 nm) before blue-shifting to generate

15Z

Po (Figure 7). If the absorption maximum provides a simplistic metric of bilin

conjugation, then the observed FR-absorbing Meta-Gf intermediate implicates planarization of the orange-absorbing Meta-Go bilin chromophore. The Meta-Gf chromophore would then adopt a twisted configuration upon conversion into the orange absorbing

15Z

Po. Since secondary

dynamics of only a few red/green CBCRs have been characterized, it remains unclear whether the monotonic spectral evolution observed in AnPixJ and Slr139330,

69

or the non-monotonic

evolution in NpF2164g7 are more representative of red/green CBCRs as a subfamily. Moreover, other members of this subfamily with comparably fast dark reversion have not been characterized on this timescale. Thus, although our data provide a benchmark for future examination of CBCR power sensors, it is not currently possible to correlate the photodynamic behavior of NpF2164g7 to its function as a power sensor.


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CONCLUDING COMMENTS NpF2164g7 dynamics are unique among CBCRs reported to date, including all characterized CBCRs within the red/green subfamily. The excited-state forward (Z → E) photodynamics are strongly inhomogeneous both spectrally and temporally and do not “homogenize” as the photocycle propagates. The excited state population de-excites via three independent and parallel evolving sub-populations (i.e., primary dynamics) that propagate down three pathways evolving in parallel (i.e., secondary dynamics) to generate

15E

Pg with high

photochemical quantum yield. The primary reverse (E → Z) dynamics of NpF2164g7 resemble those of other characterized red/green CBCRs, but the secondary dynamics exhibit clear reversible equilibration involving a newly resolved far-red intermediate not resolved previously. It will be necessary to examine other CBCR power sensors36-38 to learn whether the unusual behavior of NpF2164g7 is unique or is instead conserved in other CBCRs that have evolved away from photoswitching between two stable states.

SUPPORTING INFORMATION Supporting information for this work, including experimental setups schemes, residual comparisons for different models, sequential analyses, and comparisons of NpF2164g7 kinetics to NpF2164g3 can be found online at: http://pubs.acs.org.

ACKNOWLEDGEMENTS This work was supported by grants from the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, United States Department of


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Energy (DOE DE-FG02-09ER16117) to both J.C.L. and D.S.L. Dr. Mikas Vengris (Light Conversion Ltd.) is acknowledged for the donation of global and target analysis software.

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“Non-Canonical Photodynamics of the Orange/Green Cyanobacteriochrome Power Sensor NpF2164g7 from the PtxD Phototaxis Regulator of Nostoc punctiforme” Julia S. Kirpich, CheWei Chang, Dorte Madsen,a Sean M. Gottlieb,a Shelley S. Martin, Nathan C. Rockwell, J. Clark Lagarias, and Delmar S. Larsen


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